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Department of Animal Science, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada, Obihiro 080-8555, Japan
(Requests for offprints should be addressed to H Kuwayama; Email: fujino{at}obihiro.ac.jp)
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Abstract |
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Introduction |
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Recent studies investigating the involvement of ghrelin in ruminant physiology have reported that ghrelin immunoreactive cells are present in the abomasum of cows (Hayashida et al. 2001), and that the preprandial increase and postprandial decrease in plasma ghrelin levels are regulated by cholinergic neurons in sheep (Sugino et al. 2004). Furthermore, in vivo and in vitro studies on the effect of ghrelin on the adenohypophysial axis of ruminants have been performed by utilizing supraphysiological doses of rat or human ghrelin (Hayashida et al. 2001, Hashizume et al. 2003, 2005, Itoh et al. 2005), and it has been reported that ghrelin stimulates GH secretion in dairy cattle at different physiological stages. A very recent study using 1 µg human ghrelin/kg body weight (BW) showed that ghrelin also has different regulatory effects on plasma pancreatic hormone levels and on glucose concentration (Itoh et al. 2006). However, to our knowledge, there is no report of the dose-dependent effect of bovine ghrelin, especially of physiological doses, on central and peripheral hormones regulating energy homeostatis in ruminants. In this experiment, we validated the radioimmunoassay (RIA) systems for bovine active ghrelin and for bovine total ghrelin, which recognizes acylated ghrelin, des-acyl ghrelin and all ghrelin peptides with an intact bovine C-terminal ghrelin. We also studied the effect of intravenous injection of synthesized acylated bovine ghrelin(1–27) on plasma ghrelin, GH, insulin and metabolite levels in Holstein heifers.
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Materials and Methods |
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Animals
Six Holstein heifers born at the Field Science Center (FSC), Obihiro University, were housed under a natural light-darkness cycle. Heifers were 6 months old with an average body weight of 178.4 ± 6.4 kg (± S.E.M.) at the beginning of the experiment. They were fed twice daily (0800 h and 1600 h) with concentrates for growing calves (crude protein 14%, crude fat 1.5%, crude fiber 8%, and crude ash 7%; Hokuren, Sapporo, Japan) under the routine management practices of the FSC, Obihiro University. Animals finished eating the concentrate within 20 min. Timothy hay, mineral salt block and water were available ad libitum. Body weight was measured one day prior to each injection. At the end of the experiment, heifers had an average BW of 196.2 ± 7.8 kg and the average daily gain during the experiment was 0.89 kg.
Peptides
N-acylated bovine ghrelin(1–27) (GSSFLSPEHQKLQRKEAKKPSGRLKPR), [Cys-12]-ghrelin(1–11) with acylation and [Cys-0]-bovine ghrelin(11–27) were synthesized by manual Fmoc (9-fluorenylmethoxycarbonyl) solid phase peptide synthesis procedures. Details of the procedures for ghrelin synthesis and octanoylation on the Ser3 residue will be presented elsewhere. Peptidyl resin was cleaved with trifluoroacetic acid (TFA) followed by purification on a TSK ODS-120A column (7.8 mm ID x 30 cm) by high performance liquid chromatography (HPLC, linear gradient of 0–60% CH3CN), and fractions containing peptides were lyophilized and stored at –80 °C. Lyophilized bovine ghrelin(1–27) was dissolved in de-ionized water and diluted to 1 mg/ml with sterilized 0.1% BSA-saline for the in vivo study.
Antibodies for ghrelin RIA
Synthesized [Cys-12]-ghrelin(1–11) and [Cys-0]-bovine ghrelin(11–27) were separately conjugated to maleimide-activated mariculture keyhole limpet hemocyanin (mcKLH) and a polyclonal antibody for each antigen was generated in rabbit and guinea pig respectively. The corresponding antibody-containing serum was harvested and stored at –80 °C. Antiserum against [Cys-12]-ghrelin(1–11) only recognizes acyl-ghrelin while antiserum against [Cys-0]-bovine ghrelin(11–27) recognizes C-terminal bovine ghrelin.
Peptide injection and sampling procedures
Animals were subjected to a complete blocked Latin Square Design (six animals x six doses of injection x six days of sampling with one or two days recovery period) to obtain the minimal residual effects. For both injection of peptide and blood sampling procedures, a jugular vein catheter was inserted into each heifer one day before the start of the experiment, and the patency of the catheter was maintained with heparinized saline. Bovine ghrelin(1–27) was freshly diluted with a sterilized solution of 0.1% BSA-saline to reach the desired concentration for each animal before injection. Animals were loosely chained to the stanchion during sampling and were injected with vehicle (5 ml 0.1% BSA-saline as control) or vehicle containing synthesized bovine ghrelin (0.1, 0.5, 1.0, 5.0, 10.0 µg/kg BW) at 1100 h (3 h after the morning feed) via a jugular catheter followed immediately by flushing out of the catheter with 5 ml heparinized saline. Blood samples were collected at –30, –15, 0, 5, 10, 15, 20, 30, 45, 60, 90, 120, 150, 180 min relative to the time of injection and put into pre-ice-chilled heparinized tubes. Plasma was isolated after centrifugation (3000 r.p.m. –20 min at 4 °C) and stored at –30 °C until assay. For plasma ghrelin assay, 600 µl plasma were separately acidified with 30 µl 1 M HCl and stored at –30 °C.
RIA for bovine ghrelin, GH and insulin
Acylated bovine ghrelin(1–27) was radioiodinated by the Chloramine-T method (Tai et al. 1975) and purified by HPLC. The tracer was stored at –80 °C in 60% CH3CN/0.1% TFA and was stable for two months. This tracer ligand was used for both active and total ghrelin assays with acylated bovine ghrelin(1–27) as standard. Standards and plasma samples were incubated with the corresponding antibodies diluted in assay buffer (0.05 M sodium phosphate containing 0.25% BSA, pH 6.85). The rabbit anti-bovine ghrelin(1–11) antiserum for active ghrelin RIA and the guinea pig anti-bovine ghrelin(11–27) antiserum for total ghrelin RIA were used at final dilutions of 1:240 000 and 1:15 000 respectively. After 24 h incubation, 125I-bovine ghrelin (8000 c.p.m./100 µl assay buffer containing 1% carrier serum) was added to all tubes. Bound and free ligands were separated by the second antibody method after a further 24-h incubation, and radioactivity in the pellet was counted with a gamma counter (ARC-1000, Aloka, Japan). Triplicate standards and duplicate samples were run within a single assay at 4 °C, except samples where the concentrations exceeded the range of the total ghrelin assay. These samples were adjusted after re-assay with diluted plasma. Displacement curves of diluted bovine plasma with 125I-bovine ghrelin were parallel to the standard curve for both active and total ghrelin (Fig. 1A, B
). The average recovery rates of three known amounts of bovine ghrelin added to the bovine plasma pool were 105% and 106% for active and total ghrelin respectively. Sensitivities of the present assay were 14.2 pg/ml and 0.14 ng/ml, IC50s were 264.1 pg/ml and 8.5 ng/ml, and intra-assay coefficients of variation were 6.3% and 7.3% for active and total ghrelin RIA respectively.
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Measurements of plasma metabolites
Plasma glucose and non-esterified fatty acid (NEFA) levels were measured by commercially available kits (code nos 439–90901 and 279–75401 respectively; Wako Chemicals, Osaka, Japan).
Statistical analysis
All data are expressed as means ± S.E.M. Statistical differences between averaged pre-injected values (from –30 to 0 min) and post-injected concentrations at each time point were analyzed using one-way ANOVA, followed by multiple comparisons of general linear mixed model. Area under the curve (AUC) of each hormone for the 0 to 60 min response to injection of vehicle or ghrelin was calculated using the trapezoid method and differences were evaluated by Student’s paired t-test. All analyses were performed using SPSS for Windows, version 10.0.0 (SPSS, Chicago, IL, USA). P<0.05 was considered significant.
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Results |
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. The RIA for active ghrelin utilizing the rabbit anti-bovine ghrelin(1–11) antiserum showed that there were no differences in pre-injected plasma active ghrelin concentrations in any of the groups (159.0 ± 19.1 pg/ml). Exogenous ghrelin administration increased plasma active ghrelin levels in a dose-dependent manner (Fig. 2A). Significantly higher peak amplitude was seen 5 min after 0.5 µg/kg BW injection compared with pre-injected levels (363.8 ± 123.1 vs 195.1 ± 33.7 pg/ml, P<0.01) and increased dose-dependently. Thereafter, the elevated active ghrelin levels quickly returned to the pre-injected levels within 10 min. On the other hand, the RIA for bovine total ghrelin utilizing the antibody against bovine ghrelin(11–27) showed that the basal concentration of bovine total ghrelin was 2.8 ± 0.7 ng/ml and this value is approximately 16 times higher than the basal active ghrelin level. Total ghrelin also increased transiently following synthetic bovine ghrelin injection and returned to the pre-injected values within 5–15 min (Fig. 2B). Similar to the changes in active ghrelin levels, significantly higher peak amplitude was also seen 5 min after 0.5 µg/kg BW injection compared with pre-injected levels (24.1 ± 5.1 vs 3.9 ± 1.5 ng/ml, P<0.001) and increased dose-dependently.
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. Plasma GH concentrations in the vehicle-injected group did not change throughout the experiment (2.6 ± 0.6 ng/ml). Acylated bovine ghrelin(1–27) injection stimulated GH secretion dose-dependently. Plasma GH level peaked significantly 5 min after injection of 0.1 µg ghrelin/kg compared with pre-injected values (6.1 ± 1.9 vs 2.1 ± 0.5 ng/ml, P<0.05). The peak amplitude and duration of the GH response were dependent on the dose of ghrelin, i.e. higher doses of bovine ghrelin resulted in increased GH levels over a longer period (Fig. 3).
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. Vehicle injection did not modify the plasma insulin level throughout the study (0.7 ± 0.1 ng/ml). The plasma insulin concentration increased, in general, about 10–15 min after ghrelin administration and decreased within 45 min post-injection. Transient but significant peaks were seen only in groups injected with the higher doses of ghrelin (5.0 µg/kg BW and 10.0 µg/kg BW).
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Figure 5 shows the effect of synthetic bovine ghrelin on plasma NEFA levels in Holstein heifers. Basal NEFA concentration in the control group (35.7 ± 3.8 µEq/l) was lower than that of the ghrelin-injected groups and significant changes were not seen throughout the experiment. Injection of bovine ghrelin tended to increase plasma NEFA levels but the values did not reach significance compared with pre-injected values when ghrelin was given in low doses (0.1 µg/kg BW and 0.5 µg/kg BW). On the other hand, plasma NEFA concentration increased as early as 5–10 min after injection with the higher doses of ghrelin (from 1.0 µg/kg BW to 10.0 µg/kg BW) and returned to pre-injected values 60 min later.
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summarizes the area under the curves (AUCs) of plasma hormones and metabolites 0–60 min after ghrelin injection. Mean AUCs for six animals were pooled after subtraction of pre-injected AUCs and were expressed as a percentage of the AUC for the vehicle-injected group. Active ghrelin AUC pooled for 0–60 min started to increase significantly when the dose of ghrelin was increased to 0.5 µg/kg BW compared with that of the vehicle-injected group (11934.2 ± 1470.6 vs 9472.6 ± 1091.2 pg/ml/min). Total ghrelin AUC after ghrelin injection at 0.1 µg/kg BW was 110% of the value of the vehicle-injected group (172.2 ± 42.7 vs 155.9 ± 35.1 ng/ ml/min) and increased dose-dependently (P<0.001). Reflecting the changes in GH concentration, GH AUC for 1 h post-injection with the smallest dose of bovine ghrelin (0 .1 µg/kg) was significantly greater than that of the control group (P<0.01). The GH AUC after injection with the highest dose of ghrelin (10.0 µg/kg BW) increased 797% over that of the vehicle-injected group (1186.66 ± 16.95 vs 148.86 ± 1.43 ng/ml/min, P<0.001). However, none of the glucose AUCs 1 h after injection with several doses of ghrelin was significantly different compared with the vehicle-injected group (5221.3 ± 112.8 mg/dl/min). Moreover, insulin AUC did not increase significantly in any of the ghrelin-injected groups compared with the vehicle-injected group (44.8 ± 5.6 ng/ml/min). In all ghrelin-injected groups, pooled NEFA AUCs for 0–60 min post-injection were significantly higher than that of the vehicle-injected group (2208.9 ± 165.5 µEq/l/min).
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Discussion |
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). Therefore, we consider that the values measured with the first RIA system specifically represent the acylated ghrelin levels. On the other hand, the antibody used for the second RIA system (guinea pig anti-[Cys-0]-bovine ghrelin(11–27) serum) reacts with octanoylated bovine ghrelin(1–27) and bovine des-acyl ghrelin, but does not react with porcine acylated ghrelin, porcine des-acyl ghrelin or human acylated ghrelin (Fig. 1B), indicating that this antibody specifically recognizes the C-terminal bovine ghrelin fragment (11–27). Therefore, since the values measured using this RIA system represent acylated ghrelin, des-acyl bovine ghrelin, and all ghrelin fragments with an intact bovine C-terminal region (11–27), we use the term ‘total ghrelin’ for these values. The bovine ghrelin RIA showed that both isoforms of ghrelin (acylated and des-acyl ghrelin) are circulating in the bloodstream of Holstein heifers in considerable amounts. This result is in agreement with other reports in human and animal models (Hosoda et al. 2000a, Asakawa et al. 2005). The basal level of total ghrelin is approximately 16 times higher than that of active ghrelin, but it should be noted that this ratio can only be used for the six-month-old heifers and not for animals of other physiological stages such as pre-ruminant animals i.e. calves. In the pre-ruminant stage, ingested liquid feeds by-pass the reticulorumen and enter directly into the abomasum where ghrelin immunoreactive cells exist. After weaning, ingested dry feeds are slowly digested by ruminal microbes, and the nature of the nutrients absorbed by the abomasum become different from the pre-ruminant stage. Therefore, weaning may alter the basal plasma levels of the gastric-derived hormone, ghrelin, the secretion of which is influenced by ingested nutrients (Korbonits et al. 2004). This hypothesis is supported by a study in which basal gut hormones such as gastrin, cholecystokinin, pancreatic polypeptide and vasoactive intestinal peptide levels increased, while secretin and somatostatin levels decreased after weaning in Holstein calves (Toullec et al. 1992). Moreover, orally ingested glyceryl trioctanoate rich in milk lipids significantly increases the active ghrelin levels in murine stomach, resulting in the amount of active ghrelin in stomach tissues increasing gradually during the suckling period and decreasing sharply after the initiation of weaning; plasma total ghrelin levels do not change across this transitional period (Nishi et al. 2005). Therefore, it has been suggested that plasma active and total ghrelin concentrations and their ratio in plasma may alter with different physiological stages such as before and after weaning in ruminants.
On the other hand, the role of des-acyl ghrelin in ruminant physiology is still not known. In rat, administration of des-acyl ghrelin decreased food intake and gastric emptying rate through an action on the paraventricular nucleus and the arcuate nucleus in the hypothalamus (Asakawa et al. 2005). Des-acyl ghrelin is supposed to induce negative energy balance, in contrast to acylated ghrelin (Tschöp et al. 2000, Nakazato et al. 2001), and possesses some peripheral effects (Thompson et al. 2004, Gauna et al. 2005). Recently, Iqbal et al.(2006) reported that injection of acylated ghrelin failed to induce voluntary feed intake but significantly stimulated GH secretion in sheep, while Toshinai et al.(2006) reported that intracerebroventricular injection of des-acyl ghrelin stimulated feeding during the light phase via a distinct pathway from GHS-R in rats fed ad libitum. Therefore, it is suggested that dominant amount of circulating des-acyl ghrelin may also play an important role in mechanisms such as regulation of feeding in ruminants. Further extended studies emphasizing the effects of feeding, physiology and energy states on plasma ghrelin levels, and studies on the role of des-acyl ghrelin in ruminant physiology are necessary.
In this study, both active and total ghrelin levels were only temporarily elevated in plasma after injection of synthetic bovine ghrelin and were degraded/eliminated in the bloodstream within 15 min. This result was consistent with other reports in which the half-life of plasma ghrelin was supposed to be very short (Tschöp et al. 2000, Gauna et al. 2004). Similar results have been reported in weaned pigs; serum ghrelin levels peaked within 15 min of intravenous infusion of human ghrelin (2 µg/kg BW) and decreased to nearly half-maximal values by 30 min after infusion (Salfen et al. 2004).
In the dairy industry, it is of economical importance to increase plasma GH secretion because it is associated with faster growth, less fat stores and improved milk production (McMahon et al. 2001). Two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin (SRIF) are known to be the main regulators of episodic GH release from the anterior pituitary gland where GHRH stimulates and SRIF inhibits GH secretion. Other synthetic enkephalin derivatives, GH-releasing peptides (GHRPs, also known as GHSs) also stimulate the secretion of GH (Bowers 1998). The endogenous ligand of GHS-R, ghrelin, has been of interest as a peripheral potent GH stimulator since it was first purified from rat stomach (Kojima et al. 1999), and several studies have been published on the physiological and pharmacological effects of ghrelin in humans and in domestic animals (Korbonits et al. 2004). It has also been reported that the octanoylation on the Ser3 residue of the N-terminal region is important for ghrelin to activate its receptor, GHS-R1a (Kojima et al. 1999) and short peptides encompassing the first 4–5 residues of ghrelin can activate the GHS-R1a almost as efficiently as the full-length ghrelin (Bednarek et al. 2000). In contrast, truncated peptides of octanoylated ghrelin, such as EP-01037 (Gly-Ser-Ser(O-octanoyl)-Phe-NH2) and EP-00775 (Gly-Ser-Ser(O-octanoyl)-Phe-Leu-Ser-Pro-Glu-NH2) neither stimulate GH secretion in vivo nor replace radiolabeled ghrelin from binding sites (Torsello et al. 2002). Moreover, bovine ghrelin is a 27 amino acid peptide and 8 amino acids of the C-terminal region (11–27) are heterologous to those of monogastric animals (Dickin et al. 2004). Based on these facts, it is preferable to use a specific peptide to investigate the physiological effect of ghrelin on the mechanism of GH secretion in ruminants.
In this study, we first reported the dose-dependent stimulatory effect of synthetic acylated bovine ghrelin(1–27) on GH secretion in Holstein heifers. We used 6 doses of synthetic bovine ghrelin from 0.1 µg/kg BW to 10 µg/kg BW (approximately 0.033 nmol/kg BW to 3.3 nmol/kg BW) in single bolus injections. The minimal dose of bovine ghrelin used in this study induced significantly greater GH AUC 1 hour after injection compared with that of the vehicle-injected control group. The GH peak amplitude also reached significance as early as 5 min after injection. Itoh et al.(2005) also reported that an intravenous bolus injection of human ghrelin (0.3 nmol/kg BW, approximately equivalent to 1 µg/kg BW) significantly stimulated GH secretion in dairy cattle, and the effect seemed to be more potent in early lactating cows than in calves and in late lactation cows. In contrast, Hashizume et al.(2005) reported that intra-arterial injection of rat ghrelin at 1 µg/kg BW was not able to stimulate GH secretion in ovariectomized goats but injection of ghrelin at 3 µg/kg BW significantly stimulated GH release. Moreover, Iqbal et al.(2006) reported that a bolus injection of ovine ghrelin at a dose of 1, 5, 10 and 20 µg into the third cerebral ventricle induced a weaker GH response compared with DAP-octanoyl3 human ghrelin injection in ovariectomized ewes. These conflicting results of different responses of GH to ghrelin injection might be due to differences in experimental conditions, differences in the physiological stage of the experimental animals such as age, different preparations of peptide for injection and the use of human or rat ghrelin which have quite different amino acid residues at the C-terminal region compared with ruminant ghrelin (Dickin et al. 2004). Our results from the dose-dependent study clearly indicated that a physiological dose of bovine ghrelin is sufficient to induce GH secretion in Holstein heifers (Fig. 3 and Table 1) and this is consistent with the results of previous work in humans and rats (Takaya et al. 2000, Hataya et al. 2001, Tolle et al. 2001). Moreover, the highest dose of ghrelin used in this study (10.0 µg/kg BW) still stimulated the secretion of GH significantly more than the 5.0 µg/kg BW indicating that 10.0 µg/kg BW is not the saturation dose for the maximum GH release in Holstein heifers. Altogether, these data suggest that ghrelin is a potent GH releaser in ruminants.
The increase in plasma insulin concentration in response to synthetic bovine ghrelin injection reached significance at 10 min when the heifers were injected with 1.0 µg/kg BW while lower doses of ghrelin failed to stimulate insulin secretion. However, none of the insulin AUCs for 1 h post-injection in the ghrelin-injected groups reached significance compared with the vehicle-injected group. The effect of ghrelin on pancreatic hormones is still unclear. There are some reports suggesting that ghrelin has stimulatory (Date et al. 2002, Lee et al. 2002) or inhibitory (Broglio et al. 2001, Reimer et al. 2003) effects on insulin secretion in humans and rats. Moreover, Salehi et al.(2004) reported that a physiological dose of ghrelin has no effect on glucose-stimulated insulin release from isolated mouse islets while low doses inhibit and high doses stimulate. Furthermore, injection of 1 and 10 nmol ghrelin/kg BW suppresses basal plasma insulin levels in intact mice. A very recent study in ruminants reported that injection of 1 µg ghrelin/kg BW has a stimulatory effect on insulin in lactating cows but not in calves (Itoh et al. 2006). Combining these results with ours in which physiological doses of ghrelin failed to elevate insulin levels, it seems that physiological concentrations of ghrelin do not influence insulin secretion. Coincidentally, the injection of physiological or supra-physiological doses of ghrelin failed to induce apparent changes in plasma glucose concentrations in this study. This result is in agreement with other reports in ruminant studies on the effect of KP-102, another potent GH secretagogue (Roh et al. 1996), and ghrelin (Itoh et al. 2006). On the other hand, it was reported that injection of acyl ghrelin induces a rapid rise in insulin and glucose levels but decreases insulin sensitivity in human models (Gauna et al. 2004). These authors also reported in another in vitro study that acylated ghrelin induced while des-acyl ghrelin inhibited glucose output from primary porcine hepatocytes (Gauna et al. 2005). From this dose-dependent study, it is not possible to extrapolate the precise regulatory mechanisms of ghrelin, insulin and glucose in ruminants and more extended studies are needed in these area.
In humans, a 2-h intravenous infusion of acylated human ghrelin (7.5 pmol/kg/min) significantly increased not only serum GH concentrations, but also serum NEFA, adrenocorticotropin, epinephrine and prolactin levels when ghrelin was given at a dose of 15 pmol/kg/min (Lucidi et al. 2005). In our results in ruminants, a bolus injection of bovine ghrelin stimulated plasma NEFA levels (Fig. 5) but the response was slightly delayed compared with the time-course of ghrelin-induced GH release. This result is similar to a previous report (Roh et al. 1996) in which serial injections of KP-102 resulted in elevation of plasma NEFA levels in Holstein calves. In rodents, chronic ghrelin administration has been shown to increase body fat content and to stimulate the activity of glycerol-3-phosphate dehydrogenease and the differentiation of rat parametrial preadipocytes in vitro (Tschöp et al. 2000, Korbonits et al. 2004). These controversial effects of ghrelin on lipid metabolism might be due, at least in part, to differences in species, length (chronic and acute) and dose of peptide administration. One possible explanation for our results is that elevated plasma ghrelin may mimic the temporary negative energy balance in heifers because plasma ghrelin levels were elevated in conditions of energy insufficiency (Korbonits et al. 2004, Anderson et al. 2005). In addition, ghrelin-induced elevation of plasma GH concentration may promote fatty acid degradation (McMahon et al. 2001). As a result, reserved body fat is mobilized to maintain energy balance, leading to the subsequent increase in plasma NEFA concentration (Chilliard et al. 2000). Other factors such as leptin (adipose tissue derived protein), insulin and glucagons may participate in the counter-regulation of fat metabolism with ghrelin.
In conclusion, our dose-dependent study clearly demonstrates that both acylated and des-acyl ghrelin are circulating in the bloodstream in considerable quantities in ruminant species. Moreover, our data show that intravenous injection of ghrelin at physiological doses is sufficient to induce endogenous GH secretion and confirm the potent stimulatory effect of acylated bovine ghrelin on plasma GH secretion in Holstein heifers. Ghrelin also has considerable effects on plasma insulin and NEFA levels indicating that ghrelin plays an important role in ruminant physiology.
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Acknowledgements |
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Funding |
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References |
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Received in final form 3 March 2006
Accepted 17 March 2006
Made available online as an Accepted Preprint 22 March 2006
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—), porcine acylated ghrelin (—
—), porcine des-acyl ghrelin (—
—) and human acylated ghrelin (—
—). Inhibition of 125I-labeled bovine ghrelin binding to both antisera by serial dilution of pooled bovine plasma (
) parallel to the curve was obtained using control full-length bovine ghrelin as standard. Each point is the mean of triplicate determinations. B/BO, bound/bound in zero standard.
